Selective photocoagulation

ABSTRACT

A method of scanning a laser beam across a set of cells includes during a first interval, scanning a laser beam across a set of cells; and during a second interval, deflecting the laser beam away from the set of cells. The first interval is selected to cause microcavitation in at least a portion of the cells from the set of cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.11/428,018, filed Jun. 30, 2006, which is a continuation of U.S.application Ser. No. 10/296,417, filed Jul. 9, 2003, which is the U.S.national stage of PCT Application No. US01/17818, filed Jun. 1, 2001,which claims the benefit of U.S. Provisional Application Ser. No.60/209,010, filed Jun. 1, 2000. The contents of all the foregoingapplications are incorporated herein by this reference.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under Air Force Officeof Scientific Research Grant F49620-96-10214. The Government has certainrights in this invention.

FIELD OF INVENTION

This invention relates to methods and devices useful in laser surgicaltechniques. More particularly, the invention relates to methods ofdetermining therapeutic end points and preventing collateral damage inlaser surgical techniques.

BACKGROUND

Laser surgery has become a generally useful technique, requiringspecialized equipment and techniques. Laser surgery is indicated in thetreatment of many eye diseases. For example, lasers are used to treatthe ocular complications of diabetes. For glaucoma patients, lasers helpto control the pressure inside the eye when medications alone do notsucceed. Lasers are used to seal holes in the retina, and prevent ortreat retinal detachments. Macular degeneration is another conditionwhere lasers can sometimes help prevent vision loss. Laser surgery isalso used after cataract surgery to improve vision, if necessary.

The retinal pigment epithelium (RPE) is a single cell layer, situated inthe back of the eye behind a sensitive neuroretinal layer, with a highpigment density that can be targeted by laser irradiation. Retinal lasersurgery can be classified into techniques which rely on thermal damageto the neuroretinal layer (such as retinal welding), and those thatdesirably do not involve damage to the neuroretinal layer (such asphotocoagulative treatment of central serous retinopathy, diabeticmacular edema, and drusen).

Conventional laser photocoagulation of the retina is performed with longpulses (on the order of from about 10 to about 500 ms) generated from acontinuous wave laser, with the majority of the energy absorbed by theRPE. Heat diffusion during the long exposure to the laser pulse resultsin a relatively large zone of thermal damage, causing irreversiblethermally-induced damage of not only the RPE cells, but also thephotoreceptors and the choroicapillaris, producing scotomas (blindspots) in the treated areas.

Selective RPE photocoagulation is a recently developed therapeuticapproach that uses short (microsecond) laser pulses to, ideally, targetretinal pigment epithelial cells while not affecting adjacentphotoreceptors in the retina, as described in U.S. Pat. No. 5,302,259 toBirngruber, and U.S. Pat. No. 5,549,596 to Latina. These treatmentmethods do not produce blind spots, as does conventional laserphotocoagulation. In fact, these treatments do not produce any visiblechanges in the fundus during treatment. However, clinicians have to relyon post surgery fluorescein angiography to determine if the treatmentendpoint has been reached, a procedure that requires approximately anhour and is inconvenient for the patient.

SUMMARY

The invention results from the discovery that detection of microbubbleswithin retinal pigment epithelial (RPE) cells formed upon absorption ofpulsed laser radiation by RPE cells can be used to inhibit or preventthermal and mechanical damage to cells proximate to those undergoinglaser treatment. Thus, the invention allows substantially instantaneouscontrol over the laser dosimetry to ensure that laser energy reaches thethreshold required for RPE cell killing (a therapeutic endpoint), butavoids the administration of laser energies sufficient to damageadjacent cells, such as photoreceptors (collateral damage control).

As used herein, “microcavitation” refers to the sudden formation andcollapse of microbubbles in a liquid, events which are primarily causedby the absorption of light by chromophores in the liquid. This term alsoapplies to bubbles formed transiently by local heating. The term doesnot necessarily require pressure changes to exist.

In one aspect, the invention features a method of scanning a laser beamacross a set of cells. The method includes, during a first interval,scanning a laser beam across a set of cells; and during a secondinterval, deflecting the laser beam away from the set of cells. Thefirst interval is selected to cause microcavitation in at least aportion of the cells from the set of cells.

Some practices further include selecting the first interval to have alength that is about 40% of the length of the combined first and secondintervals.

Other practices include causing the first interval to end upon detectinga selected extent of the microcavitation.

Additional practices include those in which scanning includes scanningat a scan rate between about 0.1 to about 10 microseconds per pixel,those in which scanning includes scanning at a scan rate between about0.5 to about 7 microseconds per pixel, and those in which scanningincludes scanning at a scan rate between about 1 to about 5 microsecondsper pixel.

Other practices also include receiving a feedback signal indicative ofmicrocavitation in the set of cells. In some of these practices,scanning is carried out at least in part on the basis of the feedbacksignal.

Additional practices include those in which scanning and deflecting bothinclude modulating an acoustic-optical scanner, those in which scanningand deflecting both include rotating a polygonal mirror, those in whichscanning and deflecting both include operating a galvometric scanner,and those in which scanning and deflecting both include operating aresonance scanner.

Additional practices include those in which the fluence of the laserbeam is changed for a subsequence scan of the laser beam. Thesepractices include those in which fluence is changed in response to aparameter of light scattered from the target cells. Exemplary parametersinclude those derived from or dependent on polarization, intensity,and/or Doppler shift of light scattered from target cells. As usedherein, a parameter derived from, or dependent upon, a value of someproperty of the light includes the value itself.

In another aspect, the invention features an apparatus for scanning alaser beam across an array of cells. Such an apparatus includes a laserfor providing a laser beam; a scanner disposed in the path of the laserbeam; and a controller for causing the scanner to scan the laser beamacross a cell array at a rate selected to cause microcavitation in thecell array.

In some embodiments, the controller is configured to cause the scannerto deflect the beam away from the cell array in response to detection ofa selected extent of microcavitation within the cell array. In otherembodiments, the controller is configured to cause the scanner to scanthe laser beam across the cell array during a first interval and todeflect the laser beam away from the cell array during a secondinterval.

Other embodiments include those in which the controller is configured tocause the first interval to be about 40% of the sum of the combinedfirst and second intervals, those in which the controller is configuredto cause the scanner to scan the laser beam though an angle betweenabout 0.1 degrees and about 5 degrees, those in which the controller isconfigured to cause the scanner to scan at a rate of between about 0.1microseconds per pixel to about 10 microseconds per pixel, those inwhich the controller is configured to cause the scanner to scan at arate of between about 0.5 to about 7 microseconds per pixel, and thosein which the controller is configured to cause the scanner to scan at arate of between about 1 to about 5 microseconds per pixel.

Additional embodiments further include a detector for receiving a signalindicative of microcavitation in the cell array.

In other embodiments, the controller is configured to control thescanner at least in part on the basis of a signal indicative ofmicrocavitation in the cell array.

Embodiments also include those in which the scanner includes anacousto-optic scanner, those in which the scanner includes a polygonalmirror, those in which the scanner includes a galvometric scanner, andthose in which the scanner includes a resonance scanner.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The present invention allows selective photocoagulation to be carriedout without the need for an inconvenient post-operative determination ofa therapeutic endpoint. The present invention allows thephotocoagulation of RPE cells without complications and tissuedestruction that can occur with conventional laser retinal surgery. Thepresent invention provides an apparatus that is specifically suited fordetermination of a real-time therapeutic endpoint, and feedback based onthis determination to minimize collateral damage which can arise frommechanical and thermal damage associated with photocoagulationtherapies.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a representative laser surgery system accordingto a particular embodiment of the invention.

FIG. 2 is an oscilloscope trace of reflectivity versus time.

DETAILED DESCRIPTION

The invention is based on the optical measurement of the onset oflaser-induced cavitation and feedback to the laser source or to theoperator to control the delivered laser energy based on the measurement.The absorption of laser energy by chromophores (specificallymelanosomes) within, or proximate to, cells produces transient(lifetimes on the order of nanoseconds to microseconds) microcavitationbubbles with diameters on the order of micrometers. The bubbles arisesince the laser excitation of the chromophores can rapidly produce localheating in the immediate vicinity of the chromophores. It has beenobserved that the local heating can be intense enough to vaporize a thinlayer of liquid in intimate contact with the chromophores. Detection ofthe presence of microbubbles is a way to determine the amount of heatingcaused by laser energy. Microcavitation causes a temporary andmeasurable change in the reflectivity of the cells being irradiated.This change is used to adjust the energy of the laser source and therebyminimize damage to proximate cells that are not desirably exposed to thesame laser energies used to cause photocoagulation or thermal energiesthat can kill those proximate cells.

Selective RPE photocoagulation with the feedback of the presentinvention provides useful therapeutic outcomes. While not bound by anyparticular theory of operation, it is believed that the selectivekilling of diseased RPE cells can stimulate neighboring RPE cells toproliferate and form a new and properly functional RPE cell layer toreplace those killed by selective photocoagulation. Thus, selective RPEphotocoagulation can serve as a method of treatment for diseasesbelieved to be associated with the RPE, such as central serousretinopathy, diabetic macular edema, and drusen.

Referring to FIG. 1, a representative laser surgery system is shown.Treatment laser source 10 sends treatment laser beam 12 to dichroic beamsplitter 14. Dichroic beam splitter 14 is adapted to allow transmittanceof treatment beam 12. Treatment beam 12 is focused by focusing lens 22to impinge on mirror 24 that directs treatment beam 12 through contactlens 26 into eye 28. Scanner 64 can be used to controllably scan theelectromagnetic radiation across the cells of eye 28. The position ofscanner 64 is variable, and an illustrative example of a position isprovided in FIG. 1. Optional probe laser 30 produces probe beam 32,which is directed onto polarizing beam splitter 34, directing probe beam32 toward quarter wave plate 36. Probe beam 32 impinges on dichroic beamsplitter 14, which is adapted to reflect probe beam 32 alongsubstantially the same path as treatment beam 12. Probe beam 32 isreflected back along the same path and a fraction of probe beam 32passes through polarizing filter 34, through focusing lens 38, and intodetector 40. The probe laser and beam is optional. The interior of eye28 is illuminated by slit lamp 42. Detector 40 sends detector signal 44to discriminator 46 for determination of the presence of signal peak 48.Determination of the presence of signal peak 48 leads to signal 50 beingsent to converter 52 which either sends signal 54 to laser source 10immediately, or stores the current treatment laser energy value and,according to a multiplier value input to the converter, sends signal 54to laser source 10 when the treatment laser energy reaches a value equalto the current treatment laser energy value plus some fraction of thatvalue, determined by the multiplier value. Signal 54 can be a stop rampsignal, or can be a signal to input into controller 62, to modulate theelectromagnetic radiation.

In some embodiments, interferometer 60 can be introduced between eye 28and detector 40. In such embodiments, probe beam 32 (alternatively,treatment laser beam 12) is divided, so that a first portion of the beamimpinges eye 28, and a second portion of the beam impingesinterferometer 60. The position of interferometer 60 is variable, and anillustrative example of a position is shown in FIG. 1. Detector 40operates to detect interference between these portions of the beam, forexample frequency or intensity interferences.

Treatment Laser Source

The treatment laser source provides a treatment beam having thefollowing characteristics. The wavelength of light (that is, the energyof light) of the treatment beam is chosen to be selectively absorbed bythe target tissue. The wavelength of the light source is desirablywithin the absorption spectrum of the chromophore present within orproximate to the cell or group of cells to be treated. For example, atreatment laser that produces visible light can be used in the practiceof the invention. Visible light is generally light having a wavelengthof from about 400 nm to about 800 nm. For retinal pigment epithelialcells, preferred wavelengths for the treatment beam range from about 400nm to about 600 nm, for example, from about 450 nm to about 550 nm.

For other medical procedures which can benefit from selectivephotocoagulation, such as treatment of neural tissue by laser surgery,other lasers can be utilized. For example, if a chromophoric materialsuch as lamp black, or other laser light absorbing material, weredelivered within, or bound to the surface of, tumor cells or other cellsto be killed, the laser wavelength could be a longer wavelength lightsource tailored to the chromophoric material. The chromophore can bedelivered to be absorbed within a cell, or can be bound to the surfaceof the cell, for example, by antibodies, or by covalent or ionicbonding. Chromophores which can be used in the practice of the inventioncan be any which will produce heat upon laser irradiation sufficient tocreate microbubbles, e.g., melanin, carbon black, gold, iron oxide, andother laser phototherapy chromophores known to those of skill in theart.

Light of significantly shorter wavelength than visible light can beabsorbed directly by a wide variety of proteins, nucleotides, and manyother cellular materials that tend to be distributed throughout cellsgenerally. Thus, a treatment beam of a wavelength much shorter than 400nm, for example, below 360 nm, does not tend to selectively affect cellscontaining visible-light chromophores, and thus should be avoided.Further, a treatment beam of significantly longer wavelength thanvisible light is not particularly strongly absorbed by the chromophoresof the RPE, and therefore penetrates deeper into the choroid,effectively creating a thicker heat reservoir under the photoreceptors.This thicker heat reservoir takes longer to cool (since the cooling timeincreases as the square of the layer thickness), and releases moreenergy into the adjacent tissue. Thus, photoreceptors are more likely tobe damaged with a treatment beam of near infrared wavelength, even ifthe pulse duration is shortened.

A laser which produces pulsed light can be used in the new methods. Forexample, pulses of pulse widths less than about 10 microseconds (μs) aredesirable, for example less than about 5 μs, 1 μs, 100 nanoseconds (ns),1 ns, or 100 picoseconds (ps). Pulse width (that is, pulse duration) ofthe laser is chosen to be sufficiently short that heat conduction awayfrom the absorbing tissue to the surrounding tissue is minimized.

Alternatively, a laser that produces continuous light can be used. Insuch embodiments, the continuous light can be “chopped,” for example, byan opto-acoustic modulator, which produces pulsed light. Such a choppercan be placed immediately in front of the laser source, so as to producethe same “chopped” light in both the through and reflected beamfractions.

In some embodiments, the laser energy is delivered to particular tissueareas, even to the limit of individual cells, as a train of shortpulses. Each pulse within the train does not contain enough energy tocause mechanical disruption, but the effect of all short pulsescumulatively creates selective thermal damage at the RPE. Such trainsare characterized, in part, by a repetition rate. In particular methodsof treating tissue within the eye, the repetition rate is desirably highenough so that the pulse train be delivered to the tissue within lessthan about 1 second, so that the effects of eye movement can beminimized. On the other hand, the repetition rate is desirably not sohigh as to be substantially equivalent to continuous wave excitation,which can produce heating effects in the bulk tissue. The repetitionrate varies from about 10 Hz to about 5000 Hz, for example, from about50 Hz to about 2000 Hz, or from about 100 Hz to about 1000 Hz.

In traditional photocoagulation, with pulse widths of from about 50 to500 ms, laser-tissue interaction is well described by thermal processes;absorption of light energy by the RPE is accompanied by heat diffusionaway from the absorbing layer to the adjacent tissue, producing a zoneof thermal damage which is visible under opthalmoscopic examination as acoagulated lesion on the retina. This thermal process remains for pulsewidths down to the sub-millisecond range. For shorter pulse widths (nsand ps) on the other hand, very little thermal diffusion can take placeon the timescale of the laser pulse. The laser energy is selectivelydeposited into the melanin granules within the RPE, creating a situationin which the temperature distribution in the cell is highly nonuniform.Discrete hot spots are created within the cell, at the energy absorbinggranules, while the rest of the cell experiences little heating. Thermaldiffusion creates temperature equilibrium on the timescale ofmicroseconds after the laser pulse (for a melanosome of approximately 1μm, the thermal relaxation takes place in approximately 1 μs). Theaverage temperature of the whole cell after thermal relaxation is muchlower than the initial temperature spikes created upon excitation. RPEkilling is observed only when the laser fluence exceeds a threshold forinitiating microscopic cavitation bubble formation inside the RPE cells.Transient heating alone below bubble formation does not appear to leadto cell killing.

Microbubbles originate from explosive vaporization of a thin (less thanabout 0.1 μm) layer of fluid surrounding the individual heatedparticles. The explosive growth of microbubbles is observed within lessthan a nanosecond after the particles are irradiated with a 30picosecond laser pulse, but the bubbles are not stable. After an initialexpansion to a maximum diameter of a few micrometers, the bubblescollapse with a lifetime of about 0.1 to 1 microsecond, the lifetimebeing fluence dependent. For fluences up to a few times themicrocavitation threshold, coalescing bubbles can form from individualbubbles, and can collapse entirely within the cell, that is, the cell isnot blown apart by the microexplosion. The cell retains its shape withlittle apparent change in morphology. Laser induced microcavitation isdescribed generally in Kelly et al., “Microcavitation and cell injury inRPE cells following short-pulsed laser irradiation,” Proc. SPIE 2975(1997), and in Lin et al., “Selective Cell Killing by MicroparticleAbsorption of Pulsed Laser Radiation,” IEEE J. Sel.Topics QuantumElect., Vol. 5, No. 4, July/August 1999, pp 963-8.

At laser fluences of approximately five times the cavitation threshold,irradiated cells undergo a remarkable expansion which does not burst thecells, but distends them severely. At lower fluences, the bubbles aresmaller, and the morphology of the cells changes very little afterbubble collapse. Individual melanosomes also undergo cavitation in asimilar manner. After bubble collapse, the melanosomes can remainintact.

A train of pulses with respect to particular areas of tissue can beproduced by using a continuous wave laser and rapidly scanning the beamover the area of tissue, so that each RPE cell effectively is exposedonly to a short pulse, such as a microsecond pulse. The cells or tissueto be treated can be repetitively exposed to such scans, to simulatemultiple pulse exposures. Single pulses can produce unwanted mechanicalperturbation of the cells or tissue being treated. The desired pulsewidth and repetition rate can be obtained by proper setting of thescanning speed (pixels/second) and scanning range. Scanning ranges canbe any dimensions less than about 1000 μm× about 1000 μm, for exampleabout 300 μm× about 300 μm. The scanning fields need not be square, butcan be rectangular, or any shape convenient to scan. The scan lines neednot be contiguous. Separated scan lines can further minimize thermalbuild-up in the bulk tissue. The exact dimensions will depend on theparticular optics utilized in the surgical setup, as can be recognizedand optimized by those of skill in the art.

In some embodiments, the laser beam is scanned by an opto-acousticdeflector, which can deflect a continuous wave laser. The continuouswave laser is able to remain “on” essentially 100% of the time. Thescanning methodology can be defined by parameters of scanning speed andscan angle. Useful scanning speed can range from about 0.1 to about 10μs per pixel, for example, from about 0.5 to about 7 μs per pixel, orfrom about 1 to about 5 μs per pixel. The scan angle can range fromabout 0.1 to about 5 degrees, for example, from about 0.5 to about 2degrees.

Scanning can be carried out by a number of different scanning devices,such as two-dimensional acousto-optic deflectors (2D-AOD), galvometricscanners, rotating polygons, and resonance scanners. In someembodiments, acousto-optic deflectors are useful, because of theirspeed, linearity across the scan, and variable scan ranges, leading tomore efficient data collection than is available with some otherscanning devices. In addition, because 2D-AOD scanning uses sound wavesin a crystal, there are no moving parts. Suitable AOD scanners arecommercially available, for example, from Brimrose Corp. Suitablescanners include a two orthogonal AO crystals to scan the optical beamin x and y directions. Scanning can be carried up to 1.6 degrees oneither axis, equivalent to a scan of 480 μm by 480 μm on the fundus ofthe eye if no contact lens is used.

Desirable laser fluences for selective photocoagulation are dependent onthe detection of microcavitation, the particular pulse width for pulsedlasers or chopped beams, the wavelength of laser light employed, thetype of cell irradiated, and the concentration of chromophoreirradiated. For example, for treatment of RPE cells using 8 ns pulsewidths of 532 nm light, the treatment laser fluences which are desirablerange from about 0.08 to about 0.16 J/cm². For treatment of RPE cellsusing 3 μs pulse widths of 532 nm light, the treatment laser fluenceswhich are desirable range from about 0.22 to about 0.44 J/cm².

Particular treatment lasers which can be used in the practice of theinvention include continuous wave lasers, including gas lasers such asargon ion, krypton ion lasers adjusted to produce visible light, as wellas solid state lasers which produce visible light, such as Nd-YAGlasers. A variety of excimer-pumped or YAG laser-pumped dye lasers canalso be used to readily produce pulsed visible excitation. In someembodiments, the treatment laser source utilized is an Nd-YAG laseroperating at 532 nm.

Probe/Detection

As shown in FIG. 1, the invention can also utilize a probe source thatprovides a probe beam. The wavelength of the probe beam can vary, but itshould be recognized that generally, it is considered desirable tofilter the generally intense treatment laser beam so that it does notsaturate the detector, and that filter means are generally not extremelyselective, so that spectral information in the immediate wavelengthvicinity of the treatment beam may not be available for monitoring.Therefore, it may be preferable to use probe source which can illuminatein spectral regions somewhat removed from the treatment sourcewavelength, for example, at least about 3 nm, 5 nm, or 7 nm away.

Detection of bubbles formed with a scanning excitation laser beam can bedone with the probe beam scanned together with the excitation beam.Alternatively, the probe beam can be left stationary somewhere withinthe scanning field, for example near the center of the scanning field.In such a configuration, the stationary probe beam will detect a bubbleonly when the excitation beam imparts enough energy to the spot coveredby the probe beam to produce a bubble, giving rise to a time-dependentsignal synchronized with the scan. Alternatively, back-scattering of theexcitation beam itself can be used to monitor bubble formation. In sucha configuration, the back-scattering intensity is detected by a detectorand compared with a reference intensity generated from the excitationbeam itself. Below the bubble formation threshold, the back-scatteringsignal will be proportional to the reference intensity, with somevariation as the beam scans over the treatment area. Above the bubbleformation threshold, the back-scattering signal will be enhanced andshow much greater fluctuation due to the expansion and collapse ofbubbles. The increase in light fluctuation can be used as a signaturefor the onset of bubble formation.

The intensity of the probe beam must be sufficient to allow monitoringof the transient events within the cell or tissue of interest. Theoptical properties of the sample can dictate the intensityconsiderations for the probe source. On the other hand, the intensityshould not be so great as to independently cause heating within the cellor tissue. The adjustment of the intensity of the probe source to meetthese criteria is within the capabilities of one of ordinary skill inthe art.

The particular absorbance and reflectance properties can dictate thegeometry of the probe and detection instruments. Any geometry whichallows detection of scattered light can be used. In particularembodiments, the probe source can be used in a through-sample orreflective (back-scattering) geometry. For in vivo applications,through-sample will not generally be possible. Back-scatteringgeometries are generally more useful for in vivo treatment. In someembodiments, the geometry is a back-scattering detection of an opticalprobe beam. For example a helium neon (HeNe) laser can be focused to a10 μm diameter spot on the tissue to be treated. The probe laser powershould be adjusted to prevent heating of the tissue by the probe beam,and can be from about 0.01 to about 1 mW, for example, from about 0.05to about 1 mW, or from about 0.1 to about 1 mW.

The probe beam can be continuous wave or pulsed. If the probe beam ispulsed, and the treatment beam is scanned, the probe beam is desirablysynchronized with the treatment beam to improve signal quality.

Detection of an optical probe can be accomplished by photodiodes,photomultiplier tubes (PMT), and other similar and associated devicesknown in the art. The various advantages and capabilities of opticaldetection systems are discussed in numerous references known to those ofskill in the art. For the present purposes, important capabilities of anoptical detection system are speed and sensitivity. In particularembodiments, an avalanche-type photodiode can be used, with a confocalaperture placed in front of the opening. Bandpass filters can beemployed to substantially eliminate the signal from reaching thedetector and overloading or possibly damaging the detector.

The output of the detector is fed into a monitoring device, such as anoscilloscope, a cathode ray-type monitor, a pen recorder, or othermonitoring device. In particular embodiments, the output of the detectoris fed into a digital oscilloscope, which is synchronously triggered bythe laser source producing the excitation beam.

Methods of Treatment

The invention includes methods of treating tissue by killing cells,individually and in groups. These methods are carried out byadministering laser energy sufficient to photocoagulate the cells withinparticular tissue, or regions of tissue, while avoiding harm to adjacentor neighboring tissue, or regions of tissue. These methods involve theformation of microbubbles within the individual target cells or groupsof target cells, but without allowing heat transfer sufficient to causesignificant damage to cells proximate the target cells. Bubble formationis used as a treatment endpoint monitor. Even if bubble formation occursat a fluence above the threshold for RPE cell killing, it can still beused to mark the treatment endpoint as long as the degree of tissuedamage at this fluence is well confined to the RPE and spares thephotoreceptors.

For example, methods of treating particular cells in RPE tissue involveexercising substantially precise control over the laser dosimetryadministered. Such control is achieved by a real-time monitor thatreflects the state of affairs within the tissue being treated. Thecontrol is based on the use of microbubble detection to determine theend-point of laser therapy for target cells, and to prevent damage tocells proximate the target cells.

As a first step to carry out therapeutic treatment involving theinventive method, target cells are identified. Target cells can be anywhich can benefit from selective photocoagulation treatment. Targetcells must be able to absorb laser energy selectively, or be treated tobe able to absorb laser energy selectively (e.g., by the introduction ofa chromophore). Suitable target cells for selective photocoagulation arethose target cells which are proximate to cells which should not bephotocoagulated. For example, retinal pigment epithelial cells, whichare proximate to neuroretinal cells, are well suited for selectivephotocoagulation. Brain tumor cells, which are proximate to normallyfunctioning cells, can also be target cells. Target cells are preparedfor exposure to a treatment laser beam by positioning focusing optics,such as a contact lens for RPE cell treatment. The application of acontact lens for laser eye surgery is well known to those of skill inthe art. The treatment laser is activated to operate initially at a lowbeam intensity, for example, from about 10% to about 80%, for example,from about 25% to about 80%, or from about 50% to about 75%, of the ED₅₀threshold determined for a particular pulse width and cell type. Forexample, for selective RPE photocoagulation, the laser can be initiallyoperated at beam fluences of from about 0.008 to about 0.064 J/cm², foran 8 ns pulse width, or from about 0.022 to about 0.176 J/cm² for a 3 μspulse. The laser beam fluence can be slowly increased while monitoringparameters of the scattered treatment beam, as determined from aback-scattering geometry, for example. One useful parameter is theintensity of the treatment beam scattered from target cells. Otheruseful parameters can be polarization of light scattered from targetcells, or Doppler shifts of light scattered from target cells, whicharise due to the expansion and other movement of microbubbles.

The monitoring should be carried out so as to determine if there is anychange, for example, a positive change, in the reflectivity of thetarget cells. As used in this context, a “change” refers to a differencein signal which is detectable by a sustained change in the slope in aplot of target cell reflectance versus time, by a sustained change inrelative target cell reflectance signal as compared to a baselinereflectance, or by observation of a visually apparent peak in a plot oftarget cell reflectance versus time. In embodiments which monitorchanges in a scanning treatment beam, the background reflectance mayfluctuate as the treatment beam passes over the relatively inhomogeneoussurfaces of target cells, which can include structures such as bloodvessels, which can show changes in reflectivity even in the absence ofbubble formation. The formation of microbubbles is expected to bediscernable over this fluctuating background, so that peaks due tobubble formation may be somewhat more difficult to detect, but notprohibitively difficult. The detection of microbubbles correlates withthe laser beam energy which is referred to as ED₅₀, that is the laserdose necessary to result in death of 50% of target cells.

Upon detection of a change in reflectivity, by digital, analog, ormanual means, the treatment laser beam intensity can be immediately orsubsequently modulated, that is, by discontinuing the increase intreatment laser beam energy. In some embodiments, the detection ofmicrobubbles signifies an immediate or substantially immediate halt inthe ramp of beam intensity increase. In some embodiments, the detectionof microbubbles will cause the beam intensity to be noted, as a digitalor analog value (as a threshold value, that is ED₅₀) and the ramp ofbeam intensity will be continued until a value of ED₅₀+xED₅₀ is reached,where x is greater than zero, and less than about two.

Bubble formation can form near the end of the laser pulse, if the laserenergy is initially selected to be low relative to the bubble formationthreshold, and gradually increased to reach this threshold. Therefore,the back-scattered signal intensity should show a sudden increase nearthe end of the laser pulse if a bubble is produced. By comparing theincoming pulse shape with the scattered pulse shape, the onset of bubbleformation can be determined.

Particular diseases in the retina are associated with retinal pigmentepithelium. The RPE has as a primary function the exchange of nutrientsto and from neuroretinal and other cells. These diseases include, forexample, central serous retinopathy, diabetic macular edema, and drusen.The invention provides a means of treatment of such diseases byselective RPE photocoagulation.

EXAMPLES

The following examples illustrate certain properties and advantagesinherent in some particular embodiments of the invention.

Example 1 Ex Vivo Transient Bubble Formation

Porcine eyes of approximately 20 mm diameter were prepared 0 to 4 hoursafter enucleation. The eyes were dissected, and the vitreous wasremoved. A sheet of 1 cm² was cut out of the equatorial region of theeye and the sample was suspended in 0.9% saline solution. After 20minutes the retina could be easily peeled off. The sample was flattenedat the edges using a plastic ring. The RPE was covered with dilutedCalceinAM (Molecular Probes) 1:1000 in PBS or Dulbecos modified eaglemedium (Gibco). A cover slip was applied on top. After 20 minutes,viable cells accumulated enough fluorescent Calcein to be distinguishedfrom dead cells by fluorescence microscopy. Calcein fluorescence wasexcited at 488 nm and detected from 540 nm to 800 nm. One fluorescenceimage was taken before and a second 15-30 minutes after irradiation.Non-fluorescing cells where classified as dead. For 12 ns experiments,the sample temperature was 20° C. For 6 μs pulses, the sample was keptat 35° C. The thresholds were calculated using a PC program for probitanalyses (Cain et al., “A Comparison of Various Probit Methods forAnalyzing Yes/No Data on a Log Scale,” US Air Force ArmstrongLaboratory, AL/OE-TR-1996-0102, 1996) after Finney (“Probit Analysis,”3rd ed. London: Cambridge University Press; 1971).

A 20× objective (NA 0.42, 25 mm working distance) was used to image thecells onto a CCD camera. The spatial resolution of the setup wasapproximately 1 μm. A frequency-doubled, Nd:YAG Laser (Continuum, SEO1-2-3, λ=532 nm, 6 mm beam) was used for 12 ns irradiation. A 200 μmsection from the center of the beam was imaged on the sample to give aflat top image of 20 μm diameter. The intensity variations at the sampledue to hot spots in the beam were below 15%, as determined by afluorescing target within the area of irradiation. 6 μs Pulses werechopped from a cw frequency doubled Nd:YAG Laser (Verdi, Coherent, λ=532nm). The Gaussian-shaped spot had FWHM of 16 μm on the sample. To probethe bubble formation, the collimated beam of a diode laser (SF830S-18,Microlaser Systems, 830 nm, 1.5×2 mm beam diameter) was focused (7×10 μmFWHM) onto the RPE cell with a maximum power of 1 mW at the sample. Theaverage Nd:YAG power was 75 mW. The probe beam was switched on for lessthen 10 μs and switched off (1% power) 2-4 μs after the end of thepulse. The light was detected in a confocal geometry and also slightlyoff the optical axis to reduce back reflectance and scattering from theoptical system and from tissue layers other than the RPE. The detectorused was an avalanche photodiode (Hamamatsu C-5460), including ahigh-speed amplifier with 10 MHz bandwidth.

For 12 ns pulses, 4 samples from 4 different eyes were taken, on which atotal of 117 spots were irradiated at different fluences (40 controlswith Nd:YAG only, 77 including probe beam). The threshold for cavitationand cell death were the same, as displayed in Table 1. FLL refers to thefluence lower threshold level, FUL is the fluence upper threshold level,and Fluence is the mean of these two determinations. The # cells is thenumber of cells exposed to irradiation.

TABLE 1 12 Nanosecond Thresholds for Cavitation and Cell Death FluenceFLL Fluence FUL Fluence (mJ/cm²) (mJ/cm²) (mJ/cm²) slope # cells celldeath 71 66 75 17 77 cavitation 71 67 75 16 77 control 71 66 81 14 40

FIG. 2 is an oscilloscope trace of a reflectance signal at 1.1 timesthreshold with a minimal lifetime of 200 ns. The diode laser wasswitched on at 0.2 μs and switched off at +3.4 As to minimize sampleheating. The Nd:YAG laser was fired at 1.2 μs, which caused a detectableincrease in probe beam back scattering in a single RPE cell.

Example 2 In Vivo Treatment of RPE Tissue

A total of six eyes of three chinchilla gray rabbits are used. Therabbits are anesthetized with ketamine hydrochloride (30 mg/kg) andxylazine hydrochloride (6 mg/kg). The eyes are dilated with 1 drop ofcyclopentolate hydrochloride and 1 drop of 5% phenylephrinehydrochloride, then a −67 diopter Goldmann planoconcave lens is placedon the eye.

For laser irradiation, the output of a Q-switch, frequency doubledNd:YAG laser at a wavelength of 532 nm is used. The pulse width iscontrolled by shaping the high voltage pulse applied to the Pockel'scell while actively monitoring the intracavity energy build up. Withoutthe active feedback, the normal Q-switch output pulse width is typically250 ns with pulse energies of several mJ at a repetition rate of 500 Hz.A probe beam is provided by a HeNe laser at 0.5 mW. The back scatteredprobe beam is detected by an avalanche photodiode. The output of thedetector is fed into an oscilloscope.

Under slitlamp examination, four 100 μm marker lesions are placedoutside the corners of a designated 300 μm×300 μm treatment area, using100 ms of continuous laser exposure each (approximately 100 mW). Thenthe treatment beam is turned on, and 100 successive scans are deliveredto the treatment area. Each eye receives four such treatment spots withlaser power settings of 0.5, 1, 2, and 3 times the ED₅₀ threshold asdetermined above. All laser treatment procedures are recorded with a CCDcamera and a video tape recorder. Fundus imaging and fluoresceinangiography is performed at 1 hour after irradiation. Changes in probebeam scattering are detected and displayed on the oscilloscope. Thedetection of microbubbles is accompanied by stabilization of laserfluence at 1.5 ED₅₀. Treatment is further carried out at this fluence.

At the completion of the treatment, the animals are sacrificed withpentobarbital injection. The eyes are enucleated and processed for lightand electron microscopy examination. The total time from laser exposureto enucleation is approximately 2 hours. Each enucleated eye is fixed inphosphate-buffered 2% glutaraldehyde for 24 hours. The anterior segmentsand vitreous are removed, and the posterior eye is postfixed inphosphate-buffered 2% osmium tetroxide, dehydrated, and embedded inepoxy resin. Thick sections (approximately 1 μm) for light microscopyare stained with toluidine blue. Thin sections for electron microscopyare stained with uranyl acetate/lead acetate. Areas treated by thescanning laser are compared with control areas and with marker lesions(coagulated with continuous wave laser) for damage to thephotoreceptors, RPE, Bruch's membrane, and the choriocapillaris.Comparison is made to verify that the RPE cells are photodamaged and thephotoreceptors, Bruch's membrane, and the choriocapillaris are undamagedand viable.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the forgoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims:

1. A method of photocoagulating cells, the method comprising: during afirst interval, scanning a laser beam across a set of cells; and duringa second interval, deflecting the laser beam away from the set of cells;wherein the first interval is selected to cause microcavitation in atleast a portion of the cells from the set of cells.
 2. The method ofclaim 1, further comprising selecting the first interval to have alength that is about 40% of the length of the combined first and secondintervals.
 3. The method of claim 1, further comprising causing thefirst interval to end upon detecting a selected extent of themicrocavitation.
 4. The method of claim 1, wherein scanning comprisesscanning at a scan rate between about 0.1 to about 10 microseconds perpixel.
 5. The method of claim 4, wherein scanning comprises scanning ata scan rate between about 0.5 to about 7 microseconds per pixel.
 6. Themethod of claim 5, wherein scanning comprises scanning at a scan ratebetween about 1 to about 5 microseconds per pixel.
 7. The method ofclaim 1, further comprising receiving a feedback signal indicative ofmicrocavitation in the set of cells.
 8. The method of claim 7, whereinscanning comprises scanning at least in part on the basis of thefeedback signal.
 9. The method of claim 1, wherein scanning anddeflecting both comprise modulating an acoustic-optical scanner.
 10. Themethod of claim 1, wherein scanning and deflecting both compriserotating a polygonal mirror.
 11. The method of claim 1, wherein scanningand deflecting both comprise operating a galvometric scanner.
 12. Themethod of claim 1, wherein scanning and deflecting both compriseoperating a resonance scanner.
 13. The method of claim 1, furthercomprising changing a fluence of the laser beam for a subsequent scan ofthe beam.
 14. The method of claim 13, wherein changing the fluencecomprises changing the fluence in response to a parameter of lightscattered from the cells.
 15. The method of claim 14, wherein changingthe fluence in response to a parameter comprises changing the fluence inresponse to polarization of light scattered from the cells.
 16. Themethod of claim 14, wherein changing the fluence in response to aparameter comprises changing the fluence in response to Doppler shift oflight scattered from the cells.
 17. The method of claim 14, whereinchanging the fluence in response to a parameter comprises changing thefluence in response to the intensity of light scattered from the cells.18. A method of causing photocoagulation of cells, the method comprisingscanning a laser across a set of cells at a scanning frequency selectedto cause microcavitation in cells from a subset of the set of cells. 19.The method of claim 18, scanning a laser comprises defining a first scanline; defining a second scan line; defining a third scan line disposedbetween the first and second scan lines; scanning the laser across thefirst scan line; skipping over the third scan line; and scanning thelaser across the second scan line; whereby heat generated by scanningacross the first scan line dissipates into cells located along the thirdscan line.